Nuclear Medicine and Biology 94–95 (2021) 92–97

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Nuclear Medicine and Biology
journal homepage: www.elsevier.com/locate/nucmedbio

Assessment of drug transporters involved in the urinary secretionof [ 99mTc]dimercaptosuccinic acid
Masato Kobayashi a,⁎,1, Asuka Mizutani a, Takaki Okamoto b, Yuka Muranaka b, Kodai Nishi c, Ryuichi Nishii d,1,
Naoto Shikano e, Takeo Nakanishi f, Ikumi Tamai g, Eugenie S. Kleinerman h, Keiichi Kawai a,iaSchool of Health Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan
Division of Health Sciences, Graduate School of Medical Sciences, Kanazawa University, Kanazawa, Japan
Department of Radioisotope Medicine, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, Japand
Department of Molecular Imaging and Theranostics, National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, Chiba, Japane
Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, Ibaraki, Japanf
Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki, Japang
School of Pharmaceutical Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, JapanhDivision of Pediatrics, University of Texas M.D. Anderson Cancer Center, Houston, USAi
Biomedical Imaging Research Center, University of Fukui, Fukui, Japanbca r t i c l e

i n f oArticle history:
Received 15 November 2020
Received in revised form 21 January 2021
Accepted 25 January 2021
Keywords:[99mTc]DMSASPECTDrug transporters
Renal proximal tubulea b s t r a c t
Introduction: We clariﬁed the renal uptake and urinary secretion mechanism of [99mTc]dimercaptosuccinic acid([99mTc]DMSA) via drug transporters in renal proximal tubules.
Methods: [99mTc]DMSA was added to human embryonic kidney 293 cells expressing human multidrug and toxin
extrusion (MATE)1 and MATE2-K, carnitine/organic cation transporter (OCTN)1 and OCTN2, and organic cationtransporter (OCT)2; to Flp293 cells expressing human organic anion transporter (OAT)1 and OAT3; and to vesicles expressing P-glycoprotein (P-gp), multidrug resistance associated protein (MRP)2, MRP4, or breast cancer
resistance protein with and without probenecid (OAT inhibitor for both OATs and MRPs). Time activity curvesof [99mTc]DMSA with and without probenecid were established using LLC-PK1 cells. Biodistribution and single
photon emission computed tomography (SPECT) imaging in mice were conducted using [ 99mTc]DMSA withand without probenecid.
Results: [99mTc]DMSA uptake was signiﬁcantly higher in Flp293/OAT3 than in mock cells. Uptake via OAT3 wasinhibited by probenecid. [99mTc]DMSA uptake into vesicles that highly expressed MRP2 was signiﬁcantly higher
in adenosine triphosphate (ATP) than in adenosine monophosphate (AMP), and probenecid decreased uptake tosimilar levels as that in AMP. In the time activity curves for [99mTc]DMSA in LLC-PK1 cells, probenecid loading
inhibited accumulation from the basolateral side into LLC-PK1 cells, whereas accumulation from the apical side
into cells gradually increased. Transport of [99mTc]DMSA from both sides was low. Biodistribution and SPECT imaging studies showed that [99mTc]DMSA with probenecid loading resulted in signiﬁcantly higher accumulation inblood, heart, liver, and bladder after [99mTc]DMSA injection compared with control mice. Probenecid induced signiﬁcantly lower accumulation in the kidney after [99mTc]DMSA injection.
Conclusions: [99mTc]DMSA accumulates in renal proximal tubular epithelial cells from blood via OAT3 on thebasolateral side, and then a small volume of [99mTc]DMSA will be excreted in urine via MRP2.
Advances in knowledge: [ 99mTc]DMSA accumulates via OAT3 in renal proximal tubular epithelial cells and isslightly excreted from the cells via MRP2.
Implications for patient care: [99mTc]DMSA may be useful for measuring renal transport function with OAT3 inpatients.
© 2021 Elsevier Inc. All rights reserved.

1. Introduction
99m

Tc-labeled dimercaptosuccinic acid ([ 99mTc]DMSA), whichhighly accumulates in the kidney cortex [1], is a major renal cortical im⁎ Corresponding author at: School of Health Sciences, Institutes of Medical, Pharmaceuticaland Health Sciences, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942, Japan.
E-mail address: kobayasi@mhs.mp.kanazawa-u.ac.jp (M. Kobayashi).
1
Contributed equally.
https://doi.org/10.1016/j.nucmedbio.2021.01.004
0969-8051/© 2021 Elsevier Inc. All rights reserved.

aging agent used in the diagnosis of renal parenchymal disorders [2]. Regarding the uptake mechanism of [ 99mTc]DMSA, two main routes for[ 99mTc]DMSA in renal tubular secretion have been proposed: tubular reabsorption from the glomerular ultraﬁltrate and basolateral uptake fromplasma by peritubular extraction. Regarding the uptake of [ 99mTc]DMSA
on the apical side, [ 99mTc]DMSA is reabsorbed by megalin/cubilinmediated endocytosis from the glomerular ultraﬁltrate [3], whereas on
the basolateral side, the sodium-dependent dicarboxylate transporter

M. Kobayashi, A. Mizutani, T. Okamoto et al.

Nuclear Medicine and Biology 94–95 (2021) 92–97

NaDC-3 (SLC13A3) has been implicated in the uptake of [ 99mTc]DMSA
from peritubular capillaries into proximal tubular epithelial cells [4].
Many medicines are transported and excreted via drug transporters
including solute carrier (SLC) transporters and adenosine triphosphate(ATP)-binding cassette (ABC) transporters in the kidney. Human renal
cells express the following major drug transporters on the apical membrane: carnitine/organic cation transporter (OCTN)1 (SLC22A4), OCTN2(SLC22A5), multidrug and toxin extrusion (MATE)1 (SLC47A1), andMATE2-K (SLC47A2) as SLC transporters; and P-glycoprotein (P-gp;
ABCB1), multidrug resistance-associated protein (MRP)2 (ABCC2),
MRP4 (ABCC4), and breast cancer resistance protein (BCRP, ABCG2) as
ABC transporters [5,6]. On the other hand, the following are expressedon the basolateral membrane: organic anion transporter (OAT)1(SLC22A6), OAT3 (SLC22A8), and organic cation transporter (OCT)2(SLC22A2) as SLC transporters [5,6]. Although [ 99mTc]DMSA accumulates in the renal cortex, the relationship between [ 99mTc]DMSA andthese transporters has not been established. The purpose of this study
is to clarify renal uptake and the urinary secretion mechanism of[ 99mTc]DMSA via these SLC and ABC transporters in renal proximal
tubules.

type was incubated with [ 99mTc]DMSA (37 kBq) for 5 min (n = 4),
and cells were removed from the tissue culture plate using 0.25%
trypsin-EDTA solution (Sigma-Aldrich). Then, the radioactivity of thecells was measured using a gamma counter. For protein assays of thecells, cellular protein content was measured with a bicinchoninic acidprotein assay kit (Thermo Fisher Scientiﬁc Inc., Waltham, MA, USA)using bovine serum albumin as a standard. Uptake of [ 99mTc]DMSA in
HEK293/OCTN1, HEK293/OCTN2, HEK293/MATE1, HEK293/MATE2-K,
HEK293/OCT2, Flp293/OAT1, and Flp293/OAT3 cells was comparedwith uptake in mock HEK293 or Flp293 cells. Using assays with the
OAT and MRP inhibitor, probenecid [8–10], [ 99mTc]DMSA with andwithout 1 mM probenecid in MHBS was added to HEK293 and Flp293
cells, and uptake of [ 99mTc]DMSA was examined in Flp293/OAT3 cells.
The uptake of [ 99mTc]DMSA is shown as % injected dose (%ID)/g protein.
2.4. Uptake experiments with vesicles
After pre-incubation of vesicles for 10 min using reaction bufferconsisting of 50 mM MOPS-Tris, 70 mM KCl, and 7.5 mM MgCl2 in thekit, 37 kBq [ 99mTc]DMSA was added for 5 min to each vesicle solutionthat included ATP (n = 4), which supplies energy for ABC transporters,or adenosine monophosphate (AMP, n = 4), which does not provide energy and was used for comparison to ATP. Radioactivity of vesicles on nitrocellulose ﬁlters was measured using a γ-ray counter(AccuFLEXγ7000, Aloka, Tokyo, Japan). Uptake of [ 99mTc]DMSA in ATP
solution was compared with that in AMP solution. A higher uptake of[ 99mTc]DMSA in ATP solution than in AMP solution indicated an effectof [ 99mTc]DMSA on ABC transporters. [ 99mTc]DMSA with and without
1 mM probenecid in MHBS was added to each vesicle solution. The uptake of [ 99mTc]DMSA is shown as %ID/mg protein.

2. Materials and methods
2.1. Materials[ 99mTc]DMSA was produced using a labeling kit purchased from
FUJIFILM Toyama Chemical Co., Ltd. (Tokyo, Japan) and 99mTcO4− eluted
from a 99Mo - 99mTc generator (Nihon Medi-physics Co. Ltd., Chiba,
Japan). Radiochemical purity of [ 99mTc]DMSA was more than 95%. [ 3H]
methyl-4-phenylpyridinium was purchased from American
Radiolabeled Chemicals Inc. (St. Louis, MO, USA, 2.96 TBq/mmol), andp-[ 14C]aminohippuric acid was purchased from PerkinElmer Inc. (Waltham, MA, USA, 2.04 GBq/mmol). Probenecid was purchased from
Sigma-Aldrich (St. Louis, MO, USA).

2.5. Transport assays with LLC-PK1 cells
LLC-PK1 cells were cultured in Dulbecco's modiﬁed Eagle's medium(Sigma-Aldrich) supplemented with L-glutamine (2 mmol/l) and 10%
fetal bovine serum without antibiotics in an atmosphere of 5.0% CO2/
95.0% air at 37 °C (pH 7.4). For preparation of a cell monolayer on a micropore support, the cells were seeded at a density of 5 × 105 cells/cm2
on a polycarbonate membrane ﬁlter (growth surface area: 1.12 cm 2,
membrane pore size: 3 μm) in Transwell cell chambers. The volumesof medium inside and outside the Transwell chamber were approximately 0.5 and 1.5 ml, respectively.
Transport assays with LLC-PK1 cells were performed as described[11]. After 10-min preincubation with MHBS, [ 99mTc]DMSA was added
to the cells (n = 4) with incubation medium on the apical or basolateralside. For inhibition assays, a ﬁnal concentration of 1 mM probenecid [12]was added in MHBS with [99mTc]DMSA addition. For transport measurements, an aliquot (50 μl) of the incubation medium from the opposite
side of the cells to which [ 99mTc]DMSA had been added was obtained
at 5, 15, 30, 60, and 120 min after the injection start of [ 99mTc]DMSA incubation. At the end of the incubation, each well was rapidly washedtwice with 1 ml of ice-cold incubation medium. The cells were then solubilized in 0.5 ml 0.1 N NaOH, and radioactivity was measured using an
auto-well gamma counter (AccuFLEX ARC-7010, Hitachi, Hitachi,
Japan). The uptake of [ 99mTc]DMSA is shown as %ID/mg protein.

2.2. Human embryonic kidney (HEK)293 and Flp293 cells for SLC transporters and vesicles for ABC transporters
To investigate SLC transporters, HEK293 cells expressing OCTN1,
OCTN2, MATE1, MATE2-K, and OCT2 and Flp293 cells (derived from
HEK293 cells that stably express the human α1A-adrenoreceptor) expressing OAT1, OAT3, or the OAT3 plasmid vector alone for mock cells
were prepared as described previously [7]. Brieﬂy, HEK293 and Flp293
cells were transfected with the respective plasmid DNA and then selected with the appropriate antibiotics; HEK293/OCTN1, HEK293/
OCTN2, HEK293/MATE1, HEK293/MATE2-K, HEK293/OCT2, Flp293/
OAT1, Flp293/OAT3, and mock cells were established. All cell lines
were grown in Dulbecco's modiﬁed Eagle's medium (Wako Pure Chemical Industries Ltd., Osaka, Japan) supplemented with 10% (v/v) fetal bovine serum (Life Technologies, Carlsbad, CA, USA), 100 U/ml penicillin,and 100 μg/ml streptomycin at 37 °C in 5% CO2.
To investigate ABC transporters, vesicles (GenoMembrane Inc.,
Kanagawa, Japan) that express high levels of human P-gp, MRP2,
MRP4, and BCRP were used. Experimental kits were also purchasedfrom GenoMembrane Inc. and used for experiments with each ABC
transporter. The high quality of the kits was checked by this company.

2.6. Biological distribution of [ 99mTc]DMSA
2.3. Uptake experiments with HEK293 and Flp293 cells
All applicable institutional guidelines for the care and use of animals
at Kanazawa University were followed. All procedures performed instudies involving animals were in accordance with the ethical standardsof Kanazawa University (the Animal Care Committee of Kanazawa University, AP-122339) and were conducted in accordance with the international standards for animal welfare and institutional guidelines.
Twenty-four ddy mice (male, 6 weeks old, SLC Inc., Hamamatsu,
Japan) were housed for approximately 5–7 weeks in a 12-h light/12-h

Expression levels of SLC transporters were conﬁrmed in HEK293
cells expressing OCTN, MATE, and OCT using [ 3H]methyl-4phenylpyridinium and Flp293 cells expressing OAT using p-[ 14C]
aminohippuric acid. One day before the uptake experiments, HEK293
and Flp293 cells expressing an SLC transporter were seeded at 4 × 105
cells/well in 12-well plastic plates. Cells were pre-incubated for
10 min using modiﬁed Hank's balanced salt solution (MHBS). Each cell93

M. Kobayashi, A. Mizutani, T. Okamoto et al.

Nuclear Medicine and Biology 94–95 (2021) 92–97

3.7, PMOD Technologies LLC, Zurich, Switzerland). Coronal and sagittal
images are displayed as similar section images.

dark cycle with free access to food and water. The mice were fasted withno food overnight with water supplied ad libitum before experiments.
Then, [ 99mTc]DMSA (37.3 ± 0.6 kBq) was injected into mice via thetail vein. Mice with a clip on the penis to prevent urination weresacriﬁced at 5, 30, 60, and 120 min post-injection (n = 3 each time
point). After blood was sampled via cardiocentesis, the heart, liver, kidney, and bladder were excised. Radioactivity in weighed tissue samples
was measured using a gamma counter. Data are expressed as % injected
dose per g wet tissue (%ID/g tissue). For the loading study with probenecid, which has a high safety level in humans, ddy mice were injectedwith a mixture of [ 99mTc]DMSA and probenecid (114.1 μg/kg mouse
body weight in 100 μl saline [10,12]), and the distribution studies
were performed using the same protocol that was used for ddy mice
without probenecid loading as control mice.

2.8. Statistical analysis
Data are presented as means and standard deviation. After normality
testing using the Kolmogorov-Smirnov test, P values were calculatedusing the two-tailed paired Student's t-test for comparison between
two groups or analysis of variance and Dunnett's test, which wereused as a multiplex analysis with a population mean value, using
GraphPad Prism 7 statistical software (GraphPad Software, Inc., La
Jolla, CA, USA). A P value less than 0.01 or 0.05 was consideredsigniﬁcant.
3. Results

2.7. Single photon emission computed tomography (SPECT) imaging with
[99mTc]DMSA

Before experiments with [ 99mTc]DMSA, we conﬁrmed the functionof SLC transporters in HEK293 and Flp293 cells using 3H- or 14Clabeled substrates. Uptake of [ 99mTc]DMSA by HEK293 and Flp293
cells expressing an SLC transporter is shown in Fig. 1. [99mTc]DMSA uptake was signiﬁcantly higher in Flp293/OAT3 cells than in mock cells.
Probenecid loading decreased the uptake of [ 99mTc]DMSA in Flp293/
OAT3 cells to similar levels as that in mock cells.
Fig. 2 shows the uptake of [ 99mTc]DMSA by vesicles overexpressing
each ABC transporter. [ 99mTc]DMSA uptake into vesicles that highly
expressed MRP2 in the presence of ATP was signiﬁcantly higher thanthat in the presence of AMP. Vesicles expressing MRP4, P-gp, andBCRP did not show different levels of [ 99mTc]DMSA uptake in ATP,
AMP, or the presence of probenecid. Probenecid loading in the vesicle[ 99mTc]DMSA (60.8 ± 3.1 MBq, n = 3) or a mixture of [99mTc]DMSA
and probenecid (114.1 μg/kg mouse body weight in 100 μl saline [10,12],n = 3) was injected into the tail vein of ddy mice. SPECT acquisition wasstarted 5 min after injection, and SPECT images were obtained at
5–10 min and 60–65 min using a U-SPECT-II/CT system (MILabs,
Utrecht, The Netherlands). The data were reconstructed using the ordered subset expectation maximization method with 16 subsets andsix iterations, including no scatter and attenuation correction. The
voxel size was set to 0.8 × 0.8 × 0.8 mm3. Post-reconstruction smoothing ﬁltering was applied using a 1.0-mm Gaussian ﬁlter. Image displays
were obtained using medical image data analysis software, Pmod (ver.
250

900

200

700

800
600

Uptake (%ID/g protein)

150

500
400

100

300
200

50

100
0

0
HEK-Mockfor OCTN

OCTN1

HEK-Mockfor MATE

OCTN2

MATE-1**120

MATE-2K

900
800

100

700

80

600
500

60

400

40

300
200

20

100

0

0

HEK-Mockfor OCT

OCT2

Flp-Mockfor OAT

OAT1

OAT3

OAT3
probenecid

Fig. 1. Uptake of [99mTc]DMSA by HEK293 or Flp293 cells expressing a SLC transporter (n = 4). Before experiments with [99mTc]DMSA, we conﬁrmed the function of SLC transporters in
HEK293 and Flp293 cells using 3H- or 14C-labeled substrates. Uptake of the 3H- or 14C-labeled substrates into HEK293 or Flp293 mock cells as control cells was 112.3 ± 35.3%ID/g protein for
HEK-Mock cells for OCTN, 318.2 ± 96.2%ID/g protein for HEK-Mock cells for MATE, 66.2 ± 15.2%ID/g protein for HEK-Mock cells for OCT, and 224.2 ± 85.0%ID/g protein for Flp-Mock cells
for OAT. On the other hand, uptake of the 3H- or 14C-labeled substrates into HEK293 or Flp293 cells expressing a SLC transporter was as follows: 427 ± 48.4%ID/g protein for HEK293OCTN1, 387 ± 43.7%ID/g protein for HEK293-OCTN2, 935 ± 84.9%ID/g protein for HEK293-MATE-1, 1194 ± 123.1%ID/g protein for HEK293-MATE-2 K, 592 ± 61.1%ID/g protein for
HEK293-OCT2, 501 ± 62.1%ID/g protein for Flp293-OAT1, and 803 ± 98.8%ID/g protein for Flp293-OAT3. [99mTc]DMSA uptake was signiﬁcantly higher in Flp293/OAT3 cells than inmock cells, and was inhibited by probenecid. ⁎P < 0.01 vs. mock cells and HEK293 cells with probenecid loading.
94

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Nuclear Medicine and Biology 94–95 (2021) 92–97††

Uptake (%ID/mg protein)ratios of accumulation in kidney to blood with and without probenecidloading were 0.41 and 0.82, respectively. At 5 min, the liver-to-blood ratios with and without probenecid loading were 0.65 and 0.75, respectively. At 60 min, the average ratios of accumulation in kidney toblood with and without probenecid loading were 3.53 and 5.21, respectively, and the liver-to-blood ratios with and without probenecid loading were 0.69 and 0.75, respectively. SPECT images at 5–10 min and
60–65 min after [ 99mTc]DMSA (Fig. 4) showed that accumulation of[ 99mTc]DMSA at 5–10 min was 2.9 ± 0.8 and 1.3 ± 0.7 in the heart,
2.5 ± 0.5 and 3.2 ± 0.7 in the kidney, and 6.4 ± 2.2 and 5.0 ± 1.9%ID/
g weight in the bladder with and without probenecid loading, respectively. The average ratios of accumulation in kidney to heart with andwithout probenecid loading were 0.86 and 2.46, respectively. At
60–65 min after [ 99mTc]DMSA injection, [99mTc]DMSA showed accumulation of 1.6 ± 0.5 and 1.4 ± 0.6 in the heart, 2.1 ± 0.8 and 3.7 ± 0.9 inthe kidney, and 8.1 ± 3.1 and 7.4 ± 2.7%ID/g weight in the bladder withand without probenecid loading, respectively. The average ratios of accumulation in kidney to heart with and without probenecid loadingwere 1.31 and 2.64, respectively.

ATP

35

AMP

30

Probenecid
25
20
15
10
5
0

Control

MRP4

MRP2

P-gp

BCRP

Fig. 2. Uptake of [99mTc]DMSA by vesicles overexpressing each ABC transporter (n = 4).
[99mTc]DMSA uptake into vesicles that highly expressed MRP2 in the presence of ATP
was signiﬁcantly higher than that in the presence of AMP. Vesicles expressing MRP4, Pgp, and BCRP did not show different levels of [99mTc]DMSA uptake in ATP, AMP, or thepresence of probenecid. With probenecid loading, uptake of [99mTc]DMSA was decreased
to levels similar to that in AMP solution. †P < 0.05 vs. the presence of AMP and ATP withprobenecid loading.

4. Discussion
solution of vesicles with high expression of MRP2 in the presence of ATP
solution decreased the uptake of [ 99mTc]DMSA to similar levels as that in
AMP solution.
In the time activity curves of [ 99mTc]DMSA in LLC-PK1 cells (Fig. 3),
accumulation from the basolateral side into LLC-PK1 cells was thehighest and signiﬁcantly different than other conditions. Probenecidloading signiﬁcantly inhibited the accumulation. On the other hand, accumulation from the apical side into cells gradually increased, and was
not inhibited by probenecid. Although transport from both sides waslow, transport from the apical side to the basolateral side was slightly
higher than that from the basolateral side to the apical side.
The biological distribution (Table 1) and SPECT images (Fig. 4) ofmice injected using [ 99mTc]DMSA with or without probenecid loadingwere obtained. Regarding the biological distribution of [ 99mTc]DMSA
in normal mice (Table 1), [ 99mTc]DMSA maximally accumulated in the
blood, lung, heart, liver, and muscle at 5 min and in kidney at 60 min,and then was washed out. The accumulation in bladder gradually increased. [99mTc]DMSA with probenecid loading resulted in signiﬁcantly
higher accumulation in the blood, heart, and liver at 5 min and bladderat 5 and 30 min after [ 99mTc]DMSA injection in comparison with mice
not treated with probenecid. On the other hand, probenecid induced
signiﬁcantly lower accumulation in kidney over 5 min after [ 99mTc]
DMSA injection. At 5 min after [ 99mTc]DMSA injection, the average

In this study, accumulation and the uptake mechanism of [ 99mTc]
DMSA via drug transporters in renal tubular epithelial cells were evaluated. [99mTc]DMSA accumulates via OAT3 from the blood into renal tubular epithelial cells on the renal basolateral side (Fig. 1), and willthen be excreted into urine via MRP2 from the epithelial cells (Fig. 2).
Anion drugs are usually transported by OATs, which are SLC transporters, and MRPs, which are ABC transporters, in the cells [13]. Probenecid, an inhibitor of OATs and MRPs [12], inhibited uptake of [ 99mTc]
DMSA into Flp293/OAT3 cells and vesicles that expressed MRP2
(Figs. 1 and 2). In our previous study [14], not only technetium-99 m-labeled mercaptoacetylglycylglycylglycine ([ 99mTc]MAG3) but also
[ 99mTc]DMSA had very high afﬁnity (90% binding for [ 99mTc]MAG3
and 93% binding for [99mTc]DMSA) for plasma proteins at a typical clinical dose. In general, medicines with high afﬁnity for plasma proteins inblood are transported from the blood through the renal basolateral side
and excreted into urine. Therefore, we estimate that [99mTc]DMSA accumulates in the cells via SLC transporters on the renal basolateral side
via OAT3.
In LLC-PK1 cells, we conﬁrmed that [ 99mTc]DMSA accumulated fromthe basolateral side into the cells and that the accumulation was
inhibited by probenecid (Fig. 3a). Therefore, we conﬁrmed that
[ 99mTc]DMSA is transported via OAT3 on the basolateral membrane. Be-

(a)

(b)
3.5

80

*
3.0

60

Transport (%ID)

Cell accumulation(%ID/mg protein)

70

50

*

40

*

30

*

20

2.5
2.0
1.5
1.0
0.5

10
0

0.0
0

20

40

60

80

100

120

0

Time (min)

20

40

60

80

100

120

Time (min)From basolateral side into cells

From basolateral side to apical side

From basolateral side into cells with probenecid

From apical to basolateral side

From apical side into cells
Fig. 3. Time activity curves of cell accumulation (a) and transport (b) for [99mTc]DMSA in LLC-PK1 cells (n = 4). Accumulation from the basolateral side into LLC-PK1 cells was signiﬁcantly
higher than that with probenecid loading. From the apical side into cells, the accumulation gradually increased, but probenecid did not affect accumulation. Transport from both sides was
relatively low. ⁎P < 0.01 vs. control and probenecid loading.
95

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Nuclear Medicine and Biology 94–95 (2021) 92–97

Table 1
Biological distribution of [99mTc]DMSA in mice with and without probenecid loading.

Normal mice

Probenecid loading

Organ (%ID/g)

5 min

30 min60 min

120 min

Blood
Lung
Heart
Liver
KidneyBladder
Muscle
Blood
Lung
Heart
Liver
KidneyBladder
Muscle1.40 ± 0.21
0.80 ± 0.16
1.98 ± 0.44
1.05 ± 0.18
1.15 ± 0.25
9.82 ± 2.49
0.42 ± 0.08
1.89 ± 0.29*
0.83 ± 0.15
2.25 ± 0.41†
1.22 ± 0.16†
0.78 ± 0.27*
11.91 ± 3.11†
0.44 ± 0.09

1.01 ± 0.15
0.68 ± 0.15
1.14 ± 0.35
0.97 ± 0.23
2.08 ± 0.39
10.55 ± 3.78
0.31 ± 0.07
1.22 ± 0.31
0.70 ± 0.16
1.28 ± 0.43
0.91 ± 0.33
1.52 ± 0.28*
13.17 ± 4.52†
0.38 ± 0.08

0.87 ± 0.12
0.61 ± 0.14
1.01 ± 0.25
0.65 ± 0.18
4.53 ± 1.03
12.48 ± 3.42
0.22 ± 0.04
0.91 ± 0.22
0.65 ± 0.15
1.09 ± 0.15
0.63 ± 0.18
3.21 ± 0.42*
14.71 ± 5.56
0.28 ± 0.10

0.22 ± 0.03
0.54 ± 0.13
0.42 ± 0.05
0.11 ± 0.03
3.81 ± 0.71
14.38 ± 4.39
0.14 ± 0.04
0.28 ± 0.19
0.55 ± 0.14
0.48 ± 0.08
0.12 ± 0.04
2.97 ± 0.98†
16.81 ± 7.06
0.16 ± 0.09

%ID/g indicates percent injected dose per gram of tissue. Values are the means ± standard deviation obtained from three mice.
*P < 0.01 and †P < 0.05 compared with normal mice.

[3] and secreted via the sodium-dependent dicarboxylate transporter
NaDC-3 from peritubular capillaries on the basolateral side into proximal tubular epithelial cells [4]. Including the results of our study, because three mechanisms function for [ 99mTc]DMSA uptake into
proximal tubular epithelial cells, most injected [ 99mTc]DMSA will accumulate in the cells.
Regarding the biological distribution in mice with and without probenecid loading (Table 1), [ 99mTc]DMSA with probenecid loading provided signiﬁcantly higher accumulation in the blood, heart, liver, andbladder at 5 min after injection, whereas lower accumulation was seenin the kidney over 5 min after injection in comparison with mice without probenecid injection. Higher accumulation of [ 99mTc]DMSA in the
bladder in the presence of probenecid was due to increased radioactivityof [ 99mTc]DMSA in blood, and then [ 99mTc]DMSA will be excreted intourine by glomerular ultraﬁltration. In addition, the average ratios of accumulation in kidney or liver to blood with probenecid loading werelower than those without probenecid at early and late [ 99mTc]DMSA injection times.

cause MRP2 transports medicines from the epithelial cells into the urineon the apical side in only one direction [5,6], [ 99mTc]DMSA was also
likely to be excreted via MRP2 from the epithelial cells into the urineon the apical side. However, accumulation of [ 99mTc]DMSA in cells
through the basolateral membrane was not increased by inhibition of
MRP2 with probenecid loading because probenecid is an OAT inhibitoron the basolateral membrane and may mainly be effective on thebasolateral side where probenecid was injected. Non-radioisotopelabeled DMSA also has afﬁnity for MRP2 [15]. However, we estimatethat the afﬁnity of [99mTc]DMSA for MRP2 is lower than that of [ 99mTc]
MAG3 for ABC transporters including MRP2 [13] because transport
from the basolateral side to the apical side was low in LLC-PK1 cells(Fig. 3b). Thus, [ 99mTc]DMSA accumulates via OAT3 from the blood on
the basolateral side into proximal tubular epithelial cells, and thensome volume of [ 99mTc]DMSA will be excreted from the cells into theapical side via MRP2. Regarding other uptake mechanisms of [ 99mTc]
DMSA into epithelial cells, [ 99mTc]DMSA is reabsorbed by megalin/cubilin-mediated endocytosis from the apical side into epithelial cells

(a)

(b)

(c)

(Bq/ml)

(d)

4.0
R

L

KidneyHeart

0.0

BladderFig. 4. Representative [99mTc]DMSA SPECT imaging without (a, c) and with (b, d) probenecid loading at 5–10 min (a, b) and 60–65 min (c, d) after injection of approximately 60 MBq[99mTc]DMSA. Probenecid, an OAT inhibitor, inhibited the accumulation of [99mTc]DMSA in kidney at 5–10 min and 60–65 min after injection under isoﬂurane anesthesia, whereas
increased accumulation was shown in the heart at 5–10 min after injection.
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M. Kobayashi, A. Mizutani, T. Okamoto et al.

Nuclear Medicine and Biology 94–95 (2021) 92–97

On SPECT imaging using [ 99mTc]DMSA with probenecid loading at
5–10 min and 60–65 min after injection, accumulation of [ 99mTc]
DMSA was signiﬁcantly increased by probenecid in the heart at
5–10 min after injection, and was decreased in the kidney at 5–10 min
and 60–65 min after injection (Fig. 4). SPECT imaging showed that
OAT3 is involved in [ 99mTc]DMSA uptake from the blood into proximal
tubular epithelial cells on the basolateral side as well. Similarly, thebiodistribution results showed that the average ratios of accumulationin kidney to heart with probenecid loading were lower than those without probenecid at early and late [ 99mTc]DMSA injection times.
According to our in vitro study, [99mTc]DMSA likely accumulates via
OAT3 from the blood on the basolateral side into proximal tubular epithelial cells (Fig. 1 and 3a). [ 99mTc]DMSA has low afﬁnity for MRP2
(Fig. 2). In the in vivo study, we also conﬁrmed that the uptake mechanism of [ 99mTc]DMSA involves OAT3 because accumulation of [ 99mTc]
DMSA in the kidney was lower in the presence of probenecid comparedwith the absence. Yee et al. showed no signiﬁcant difference in [ 99mTc]
DMSA uptake in control vs. probenecid-treated rats [16]. They injectedprobenecid into rats at 1 h before [ 99mTc]DMSA injection. With [ 99mTc]
DMSA experiments, probenecid has little effect because the competitive
inhibition effect of probenecid was provided at about 30 min in ourin vivo experiments. In their study, probenecid could not play a signiﬁcant role in renal uptake of [99mTc]DMSA. Although rodents and humans
express OAT3 [5,6] and MRP2 [17] in kidney, assessment of the expression levels of renal transporters including OAT3 and MRP2 may be important to emphasize the uptake mechanism of [ 99mTc]DMSA via renaltransporters.

18K07747) and Network-type Joint Usage/Research Center for Radiation Disaster Medical Science.

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5. Conclusion[ 99mTc]DMSA accumulates from the blood into renal proximal tubular epithelial cells via OAT3 on the basolateral side, and then a small volume of [ 99mTc]DMSA will be excreted via MRP2 in urine.
Declaration of competing interest
There is no conﬂict of interest.
Acknowledgments
The authors would like to thank Mikie Ohtake and other staff of the
School of Health Sciences, Kanazawa University. This study was fundedin part by a Grant-in-Aid for Scientiﬁc Research from the Japan Society
for the Promotion of Science (No. 18K07747) and Network-type Joint
Usage/Research Center for Radiation Disaster Medical Science.
Financial support
This study was funded in part by a Grant-in-Aid for Scientiﬁc Research from the Japan Society for the Promotion of Science (No.

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